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Characterization of the human skeletal muscle glycogen synthase gene (GYS1)promoter.

Fredriksson, Jenny; Ridderstråle, Martin; Groop, Leif; Orho-Melander, Marju

Published in:European Journal of Clinical Investigation

DOI:10.1111/j.1365-2362.2004.01299.x

2004

Link to publication

Citation for published version (APA):Fredriksson, J., Ridderstråle, M., Groop, L., & Orho-Melander, M. (2004). Characterization of the human skeletalmuscle glycogen synthase gene (GYS1) promoter. European Journal of Clinical Investigation, 34(2), 113-121.https://doi.org/10.1111/j.1365-2362.2004.01299.x

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European Journal of Clinical Investigation

(2004)

34

, 113–121

© 2004 Blackwell Publishing Ltd

Blackwell Publishing, Ltd.

Characterization of the human skeletal muscle glycogen synthase gene (

GYS1

) promoter

J. Fredriksson, M. Ridderstråle, L. Groop and M. Orho-Melander

Malmö University Hospital, Malmö, Sweden

Abstract Background

Impaired activation of the human skeletal muscle glycogen synthase by insulinis typical for type 2 diabetic patients. Regulation of glycogen synthase occurs mainly byphosphorylation/dephoshorylation but little is known whether there also is transcriptionalregulation. Therefore we studied transcriptional regulation of the human skeletal muscleglycogen synthase gene (

GYS1

) and evaluated the effects of insulin and forskolin on thepromoter activity.

Methods

Seven promoter fragments were expressed in C2C12 myoblasts and myotubes andin HEK293 cells, and the luciferase assay was used to determine transcriptional activity.

Results

The highest luciferase activity, 350-fold of the promoterless vector, was obtainedwith nucleotides

−

692 to +59 in myotubes (

P <

0·001), while the nucleotides

−

250 to +59provided the highest, 45-fold, activity in the HEK293 cells (

P <

0·001). Longer promoterconstructs (nucleotides

−

971,

−

1707 and

−

2158 to +59, respectively) had low promoteractivity in both cell types. Forskolin treatment for 24 h resulted in approximately 30%decreased promoter activity in myotubes (

P <

0·05). Insulin treatment for 0·5–3 h did notincrease

GYS1

promoter activity; instead the activity was slightly but significantly decreasedafter 24 h in myotubes (

P <

0·005).

Conclusions

From our results we conclude that basal

GYS1

promoter activity is obtainedfrom the first 250 nucleotides of the promoter, while the nucleotides

−

692 to

−

544 seem tobe responsible for muscle-specific expression, and nucleotides

−

971 to

−

692 for negativeregulation. In myotubes, the

GYS1

promoter was sensitive to negative regulation by forskolin,whereas insulin did not increase

GYS1

transcription.

Keywords

cAMP, glycogen synthase, insulin, promoter activity, transcription.

Eur J Clin Invest 2004; 34 (2): 113–121

Introduction

Glycogen synthase has a central role in glucose metabolismand exists in two isoforms. While the liver isoenzyme isexclusively expressed in the liver [1], the skeletal muscleisoenzyme is expressed in a variety of tissues besides skeletalmuscle, including at least fat, heart, kidney and brain, but

not in the liver [2]. The almost ubiquitous expression of theskeletal muscle glycogen synthase gene (

GYS1

) suggeststhat

GYS1

might exert vital housekeeping functions formost cells. Less is, however, known about the regulation of

GYS1

expression. The activity of glycogen synthase is undercomplex regulation. Glycogen synthase is inactivated byphosphorylation at several sites, and activation occurs bydephosphorylation by protein phosphatase 1 (PP1

G

) [3]. Arise in the intramuscular glucose-6-phosphate concentrationallows binding of this metabolite to glycogen synthaseand facilitates dephosphorylation by allosteric mechanisms[4]. Activation of skeletal muscle glycogen synthase is trig-gered in response to insulin stimulation, whereas adrenalinepromotes inactivation of the enzyme [4].

Activation of glycogen synthase by insulin is impaired inpatients with type 2 diabetes [5–8] and in subjects withincreased risk for developing the disease, i.e. first-degree rela-tives of type 2 diabetic patients [6], suggesting that impaired

Department of Endocrinology, Lund University, Wallenberg Laboratory, Malmö University Hospital, Malmö, Sweden (J. Fredriksson, M. Ridderstråle, L. Groop, M. Orho-Melander).

Correspondence to: Jenny Fredrikssson, Department of Endocrinology, Lund University, Wallenberg laboratory, floor 3, Malmö University Hospital, S-20502 Malmö, Sweden. Tel.: +46–40–337 214; fax: +46–40–337 042; e-mail: jenny.fredriksson@endo.mas.lu.se

Received 7 October 2003; accepted 12 December 2003

114

J. Fredriksson

et al.

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European Journal of Clinical Investigation

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glycogen synthase activity might be an inherited feature.The muscle glycogen synthase gene (

GYS1

) has been con-sidered a candidate gene for type 2 diabetes but also for themetabolic syndrome and hypertension. Associations betweenthe

GYS1

gene and type 2 diabetes has been identified inseveral populations [9–14] but no common functionalvariations have been found to explain these associations[11,14–18]. Decreased

GYS1

mRNA levels in the skeletalmuscle of type 2 diabetic patients compared with healthycontrol subjects has been reported in some [19–21] but notall studies [8,14,22]. Whether or not the expression of

GYS1

is influenced by insulin also remains to be established, asresults from different studies are divergent [8,19–25].

A prerequisite for the study of transcriptional regulationof

GYS1

is better knowledge about the promoter structure.Our aim here was to characterize the promoter region of

GYS1

and to provide information about transcriptionalregulation of

GYS1

in skeletal muscle.

Materials and methods

Sequencing of the

GYS1

5′′′′

-flanking region

A chromosome 19 clone (AC008687) containing part of the

GYS1

gene and greater than 4 kb of the 5

′

-flanking regionwas used for primer design. The sequence was confirmedby automated sequencing of human genomic DNA usinga Thermo Sequenase II dye terminator cycle sequencingpremix kit (Amersham Pharmacia Biotech, Uppsala,Sweden) in an ABI 377 (Applied Biosystems, Foster City, CA)according to the manufacturer’s instructions. Sequences wereanalyzed using Sequencher 3·0 (Gene Codes Corporation,Ann Arbor, MI) software. Putative transcription factorbinding sites were predicted using the MatInspector pro-fessional 3·5 program [26], and insulin responsive elementswere predicted according to O’Brien

et al

. [27].

Rapid amplification of cDNA ends

The Marathon Ready cDNA kit (Clontech, Palo Alto, CA)containing cDNA from human skeletal muscle was used toconfirm the previously determined transcription initiation siteof

GYS1

[11,18]. The amplified PCR products were sub-cloned into a pGEM-T vector (Promega, Madison, WI) and20 clones were sequenced with universal primers SP6 and T7.

Preparation of luciferase reporter constructs

Four fragments of the

GYS1

promoter region (containingnt

−

250,

−

995,

−

1707, and

−

2158 to nt +59) were amplifiedby PCR. Primers introduced

MluI

(at nt positions

−

995,

−

1707 and

−

2158) or

SacI

(at position

−

250) restriction sitesin the 5

′

-end of the constructs, whereas an endogenous 3-prime

HindIII

site at nt position +59 defined the end of eachpromoter construct. By digestion of the fragment containing

nt

−

995 to +59 with

XhoI

(MBI Fermentas, Vilnius,Lithuania),

BglII

(Amersham Pharmacia Biotech) or

SmaI

(Amersham Pharmacia Biotech); three shorter fragments wereobtained containing nt

−

692,

−

544 and

−

121 to +59, respec-tively. Consequently, after digestion with

HindIII

(MBIFermentas), each construct contained 59 bp of the 5

′

-untranslated region according to the previously publishedtranscription start site [11,18]. Digested and gel-purifiedpromoter fragments were then cloned into a similarlydigested pGL3-Basic vector (Promega)

.

Cell culturing

Mouse C2C12 myoblasts, a cell line that differentiates andfuses into multinucleated myotubes and is sensitive toinsulin [28], and human embryonic kidney HEK293 cells, acell line chosen to represent nonmuscle

GYS1

expression[29], were obtained from American Type Culture Collection(ATCC, Manassas, VA). Cells were maintained in Dulbecco’smodified Eagle’s medium (DMEM, ICN, Costa Mesa,CA), supplemented with 10% fetal bovine serum, 1%

-glutamine and 2% penicillin-streptomycin. The glucoseconcentration of the medium was 25 mM. To induce dif-ferentiation of C2C12 myoblasts into myotubes, mediumwas switched to DMEM containing 10% horse serum. Cellswere cultured at 37

°

C under 5% CO

2

.

Transfection assays

The day before transfection, cells were seeded into six-welltissue culture plates at 2·5

×

10

5

(HEK293), 2·8

×

10

5

(C2C12 myoblasts) or 3·8

×

10

5

(for C2C12 myotube dif-ferentiation) cells per well, respectively. Transfections wereperformed with LipofectAMINE PLUS reagent (Invitro-gen, Lidingö, Sweden) according to the manufacturer’sinstructions. Each transfection was performed using 1

µ

g ofluciferase reporter construct DNA and 100 ng of an internalcontrol plasmid pRL-TK (Promega). Three hours after thestart of transfection, fetal bovine serum was added to a finalconcentration of 10% and incubation was continued.

Luciferase assays

Luciferase assays (Dual Luciferase Reporter assay System,Promega) were carried out according to the manufacturer’sinstructions. Firefly luciferase activities were normalizedby

Renilla

luciferase activities. At least three independentexperiments including three transfections for each constructwere performed.

Treatment with forskolin or insulin

After transfection the cells were cultured for 18–24 h(HEK293 cells) or 96 h (C2C12 differentiating myotubes).Cells were serum-starved for at least 2 h before the medium

Human muscle glycogen synthase gene promoter

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© 2004 Blackwell Publishing Ltd,

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was replaced with medium containing either 10

µ

forskolin (Sigma-Aldrich, Stockholm, Sweden), 100 n

human insulin (a kind gift from Novo Nordisk, Bagsværd,Denmark), a frequently used concentration known tobe sufficient to activate insulin signalling pathways inC2C12 cells [30], or vehicle, and incubation was continuedfor 4 h and 24 h with forskolin and 0·5 h, 1 h, 3 h and 24 hwith insulin.

Statistical analysis

Data are expressed as mean

±

SEM. The significance of dif-ferences between promoter activities of the constructs weredetermined by the Mann–Whitney independent rank sumtest using the BMDP statistical software (Version 1·12,Biomedical Data Processing Statistical Software Inc., LosAngeles, CA).

P

-values less than 0·05 were consideredstatistically significant.

Results

GYS1

5′′′′

-region

DNA from a healthy subject was sequenced to confirm thesequence of

GYS1

obtained from the database. Two differ-ences were found between the obtained sequence and theAC008687 sequence. At nucleotid position

−

1464 thedatabase sequence lacked a t and had an extra t at position

−

1522 relative to the transcription initiation site. Promoter

analysis revealed several putative transcription factorbinding sites, including at least nine putative binding sitesfor the general transcription factor Sp1, or GC boxes, threeactivator protein 2 (Ap-2) sites, three cyclic AMP (cAMP)responsive elements (CRE), four sites for CAAT/enhancerbinding proteins (CEBP), two muscle initiator sequences(MINI), five octamer binding protein 1 (Oct1) sites, threesterol responsive elements (SRE), 15 E-boxes of which twowere putative sites for the myoblast determining factor MyoD,eight Ets-like motifs, two insulin response element A(IRE-A)-like motifs, and one site each for myocyte enhancerfactor 2 (MEF2)-, transcriptional enhancer factor 1- andphosphoenolpyruvate carboxykinase (PEPCK)-like motifs(Fig. 1). No TATA or CAAT boxes could be identified, althougha sequence related to a muscle TATA box was located threenucleotides downstream from the ATG start codon.

For determination of transcription initiation sites, 22 clonescontaining 5

′

-rapid amplification of cDNA ends (RACE)products were sequenced. A total of 13 putative transcriptioninitiation sites were identified (Fig. 1). The site at position

−

180 relative to the ATG codon was indicated by sequencesfrom seven different clones (Fig. 1).

Functional characterization of the

GYS1

promoter

Compared with the promoterless luciferase vector, theshortest (nt

−

121 to +59) and longest (nt

−

2158 to +59)

GYS1

promoter fragments had 4- and 10-fold higheractivities in HEK293 cells, 19- and 4-fold higher activities inmyoblasts and 28- and 45-fold higher activities in myotubes,respectively (Fig. 2).

Figure 1 Glycogen synthase gene (GYS1) promoter constructs and putative transcription factor-binding sites within the constructs. Filled triangles (�) indicate 5′-ends of the different constructs. The −250, −971, −1707 and −2158 constructs were made by introducing a MluI site in a linker primer, whereas the −692, −544, and −121 constructs were created using internal restriction sites. All constructs contained 59 bp of the 5′-untranslated region (�) numbered according to the previously determined transcription initiation site [11,18]. The major transcriptional initiation site, proposed by seven different clones in the present study, is indicated at position −14 relative to the previously determined single initiation site. Four sites, recognized by one clone each, are not shown in the figure. These sites were located at nt +92, +108, +125 and +168. Ap-2, activator protein 2; CEBPB, CAAT/enhancer-binding protein beta; CRE, cAMP responsive element; IRE-A, insulin response element A; MEF2, myocyte enhancer factor; MINI, muscle initiator; PEPCK, phosphoenolpyruvate carboxykinase; SREBP, sterol responsive element binding protein; TEF-1, transcriptional enhancer factor 1.

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© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 113–121

In HEK293 cells, the construct containing sequencesfrom nt −250 to +59 had the highest promoter activity(45-fold of the promoterless vector) and was 12-fold moreactive compared with the construct containing nt −121to +59 (P < 0·001) (Fig. 2a). The promoter activities of thefour longest promoter sequences (GYS1-692, GYS1-971,GYS1-1707, and GYS1-2158) were 0·8-, 0·3-, 0·3- and0·2-fold compared with the activity of the GYS1-250construct (P < 0·05 for the GYS1-692 construct andP < 0·001 for the GYS1-971, GYS1-1707, and GYS1-2158constructs).

In C2C12 myoblasts, a gradual increase of the promoteractivity was seen with increasing length of the promotersequence, up to the construct containing the sequencecorresponding to nt −692 to +59, which accounted for thehighest, 65-fold, promoter activity (Fig. 2b). The sequencebetween nt −692 and −250 was responsible for a twofoldincrease in promoter activity compared with the sequencecorresponding to nt −250 to +59 (P < 0·005). In contrast,a decrease in promoter activity was associated with sequencesbetween nt −971 and −692 (89% decrease, P < 0·001) andeven more between nt −2158 to −1707, leading to a totaldecrease of 94% from GYS1-692 to GYS1-2158 (P < 0·001)(Fig. 2b).

Similar to results in C2C12 myoblasts, the sequencebetween nt −692 and +59 accounted for the highestpromoter activity in C2C12 myotubes (340-fold comparedwith the promoterless vector) (Fig. 2c). The sequence betweennt −692 and −544 accounted for a threefold increase inpromoter activity (GYS1-544 vs. GYS1-692, P = 0·0014),whereas, again, the sequence between nt −971 and −692accounted for a 76% decrease (GYS1-971 vs. GYS1-692,P < 0·001). The GYS1 promoter was significantly moreactive in the differentiating C2C12 myotubes comparedwith the C2C12 myoblasts (twofold difference for theGYS1-692 construct, P < 0·005).

Effect of forskolin

Treatment of C2C12-differentiating myotubes for 4 h withforskolin resulted in decreased activities of 18% and 22%for the constructs containing sequences corresponding tont −692 to +59 and nt −1707 to +59, respectively (P < 0·05for GYS1-692 and P < 0·005 for GYS1-1707) (Fig. 3a).After 24 h of forskolin treatment, down-regulation of thepromoter activities was even more pronounced: 30%(P < 0·05) and 36% (P < 0·005) for GYS1-692 and GYS1-1707, respectively (Fig. 3b). In addition, forskolin causeda significant 46% decrease of the promoter activity carriedby the sequence corresponding to nt −121 to +59 (P < 0·001).However, as the nontreated promoter activity of the GYS1-121 construct was only 2% and 4% of that of GYS1-692and GYS1-1707, respectively, the decrease in relative luci-ferase activity of GYS1-121 was only 3% and 5% of thedecrease recorded for the two longer fragments. In contrast,forskolin treatment had no effect on GYS1 promoter activityin HEK293 cells (Fig. 3c,d).

Effect of insulin

Insulin did not exert a significant effect on GYS1 promoteractivity after 0·5, 1 and 3 h of treatment, neither in HEK293cells (Fig. 4) nor in C2C12-differentiating myotubes (Fig. 5).However, after insulin treatment for 24 h, a slight but sig-nificant decrease in promoter activity, was seen in the C2C12myotubes (Fig. 5d) for the sequences corresponding tont −250 to +59 (41% decrease, P < 0·005) and nt −2158to +59 (22% decrease, P < 0·05). Although the largest

Figure 2 Transient expression of glycogen synthase gene (GYS1) promoter constructs. HEK293 cells (a), C2C12 myoblasts (b) or C2C12 myotubes (c) were cotransfected with the seven GYS1 promoter-luciferase constructs or the promoterless pGL3-basic vector together with the pRL-TK Renilla vector. Firefly luciferase activities were corrected with the Renilla luciferase activities to adjust for transfection efficiency. Values are means ± SEM from three to eight independent experiments, each run in triplicate.

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Figure 3 Glycogen synthase gene (GYS1) promoter activity after forskolin treatment of C2C12-differentiating myotubes (a,b) and HEK293 cells (c,d), transfected with GYS1-121, GYS1-250, GYS1-692 or GYS1-1707 constructs together with the pRL-TK Renilla vector. After 96 h (C2C12) or 24 h (HEK293) following the transfection, cells were serum starved and incubated with 10 µ forskolin for 4 h and 24 h. Filled bars represent control cells and open bars forskolin-treated cells. Values are expressed as relative luciferase activities (means ± SEM) and are from three independent transfections in triplicates. *P < 0·05.

Figure 4 Glycogen synthase gene (GYS1) promoter activity after insulin treatment of HEK293 cells cotransfected with GYS1 constructs GYS1-250, GYS1-692 or GYS1-2158 together with the pRL-TK vector. After 24 h following the transfection, cells were serum starved and treated with insulin for 0·5 h (a), 1 h (b), 3 h (c) or 24 h (d). Filled bars represent control cells and open bars insulin-treated cells. Values are expressed as relative luciferase activities (mean ± SEM) and are from three independent experiments in triplicates.

118 J. Fredriksson et al.

© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 113–121

decrease in luciferase activity, calculated as relativeluciferase units, was measured for the GYS1-692 construct,this decrease of 32% did not reach statistical significance(P = 0·10).

Discussion

In this study we have functionally characterized the pro-moter of the human GYS1 gene and evaluated the effect ofcAMP as well as short- and long-term effects of insulin onthe transcriptional activity of this promoter.

Fifty-four per cent of the clones in the 5′-RACE experi-ments proposed the nucleotide at position −180 relative tothe translation start codon as the major transcriptioninitiation site. Although some of the shorter clones couldrepresent degraded mRNA products, instead of biologicaltranscription initiation sites, the finding of rare clones exten-ding upstream of position −180 indicates some variationin the transcription initiation of GYS1. The discrepancywith previous results suggesting a transciption initiation siteat −166 [11,18] could be explained either by instability ofthe 5′-part of the GYS1 mRNA, the quality of RNA usedto create the cDNA kit, or by factors forcing reverse trans-criptase to terminate such as extensive GC-rich regions orsecondary structures. Further support for the nt −180representing the major transcription initiation site is pro-

vided by the sequence surrounding the nt −180, TCC+1TTCT,which shows conservation to the reported consensus trans-cription initiation sequence [31].

We performed characterization of the GYS1 promoter inthe embryonal kidney cell line HEK293 and in C2C12myoblasts and myotubes to identify the minimal promoterof GYS1 as well as regions responsible for muscle-specificup- or down-regulation of the gene. Our results indicate thatthe first 250 bp of the 5′-flanking region is responsible forthe highest activity in HEK293 and thus accounts for thebasal GYS1 promoter activity. Lack of classical TATA-and CAAT-boxes within the GYS1 promoter, increased GCcontent and CpG islands close to the transcription initiationsite, as well as several Sp1 and Oct1 sites are all features ofa housekeeping gene [32]. This is also in line with the factthat GYS1 is ubiquitously expressed in many tissues.Despite this, GYS1 has its major role in the skeletal musclewhere glycogen is continuously synthesized and brokendown. Correspondingly, several putative muscle-specifictranscription factor binding sites were found within theGYS1 promoter. Muscle-specific gene expression as well asmyogenesis is regulated by association between myogenicbasic helix-loop-helix (bHLH) proteins like MyoD andMEF2 factors [33]. Similar to GYS1, the glycogenin 1 pro-moter harbours several E-boxes as well as binding sites forMEF2, Ap-2, Oct1 and Sp1 [34]. To establish which ofthese sites are important for the regulation of GYS1transcription, mutational analysis would be required.

Figure 5 Glycogen synthase gene (GYS1) promoter activity after insulin treatment of C2C12 myotubes cotransfected with GYS1 constructs GYS1-250, GYS1-692 or GYS1-2158 together with the pRL-TK vector. After 96 h following the transfection, cells were serum starved and treated with insulin for 0·5 h (a), 1 h (b), 3 h (c) or 24 h (d). Filled bars represent control cells and open bars insulin-treated cells. Values are expressed as relative luciferase activities (mean ± SEM) and the results are from three independent experiments in triplicates. *P < 0·05.

Human muscle glycogen synthase gene promoter 119

© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 113–121

In the C2C12 cells, the highest GYS1 promoter activity wasobserved for the sequence corresponding to nt −692 to +59,especially in differentiating myotubes where the activityof the GYS1-692 construct was threefold higher comparedwith the construct lacking this region (GYS1-544).These results suggest that the region between nt −692and −544 could be responsible for myotube-specificexpression. Sequence analysis did not provide any expla-nation for the high muscle-specific transcription from thisregion, as motifs in this region also are found throughoutthe GYS1 promoter. The region upstream of nt −692showed relatively weak promoter activity in all three celltypes, suggesting that, in particular, the region from nt−971 to −692 could be responsible for negative regulation.However, no cAMP responsive elements or other obviousbinding sites for negative regulation were found withinthis region.

Muscle glycogen synthase is subject to post-translationalregulation in response to several hormones. Insulin is knownto promote dephosphorylation and thereby activation ofglycogen synthase by initiating a cascade of events leading toinactivation of glycogen synthase kinase 3 (GSK-3), as well asto activation of protein PP1G [3]. Adrenaline has a negativeeffect on glycogen synthase, as elevation of cAMP levelspromotes both dissociation of glycogen synthase from theglycogen targeting subunit of PP1 (GM) [35] and activationof the cAMP-dependent protein kinase, which is capable ofinactivating glycogen synthase [36]. In HEK293 cells,forskolin treatment had no effect on GYS1 promoter activity,whereas in C2C12 myoblasts an inhibitory effect was seenafter both 4 h and 24 h. The negative effect of forskolin wasexpected, as the GYS1 promoter contains several putativebinding sites for transcription factors of the cAMP respon-sive element binding (CREB) family. In agreement with ourresults suggesting a role for cAMP as a negative regulatorof glycogen synthesis at the level of GYS1 transcription, thecAMP analogue db-cAMP has previously been shown tostimulate transcription of the muscle glycogen phosphory-lase promoter [37]. In addition, raised cAMP levels down-regulated transcriptional activity of the glycogenin promoter[38]. It is therefore possible that the effect of enhancedlipolysis on glycogen synthesis also involves transcriptionalsteps.

It is well established that insulin plays a crucial role inthe activation of glycogen synthase, while the effect ofinsulin on GYS1 transcription is more unclear despite beingaddressed in several studies. Although insulin was found tostimulate GYS1 mRNA expression in human skeletalmuscle biopsies in one study [25], several other studies havenot found an effect [8,19,20,22,23]. Neither could insulinsignificantly stimulate GYS1 transcription in in vitro studiesof cultured human nondiabetic myoblasts [24], while incultured diabetic myoblasts hyperinsulinaemia increased andthen normalized GYS1 mRNA to levels seen in nondiabeticcultures in normal medium [21]. Our results are compatiblewith the view that short-term treatment with insulin doesnot increase GYS1 transcription in vitro. On the contrary,we found a slightly but significantly decreased GYS1promoter activity after 24 h of treatment of cells with

insulin. It remains, however, to be established whether thisdecreased transcriptional activity is a reflection of thesituation seen in type 2 diabetes [19,20].

At least eight distinct consensus insulin-response sequenceshave been defined including the Ets- and E-box motifs,which mediate stimulatory effects, and PEPCK-like motifs,which mediate inhibitory effects of insulin [27]. In summary,we present evidence that basal GYS1 promoter activityis obtained from the first 250 nt upstream of the GYS1transcription initiation site while a region between nt −692and −544 is responsible for high muscle-specific expressionand nt −971 and −692 for negative regulation. In differen-tiating myotubes, the GYS1 promoter is sensitive to down-regulation by cAMP, indicating that epinephrine couldregulate skeletal muscle glycogen metabolism also at thelevel of GYS1 transcription. In contrast, insulin treatment didnot increase GYS1 promoter activity during these experi-mental conditions, suggesting that the effect of insulin isprimarily mediated by phosphorylation/dephosphorylationand not by transcriptional regulation.

Acknowledgements

This work was supported by grants from the Foundationfor Strategic Research through The National Network forCardiovascular Research, The Swedish Foundation for theStudy of Diabetes, the Albert Påhlssons Foundation, theSwedish Medical Research Council, Malmö UniversityHospital Foundation, the Ernhold Lundström Foundation,the Anna-Lisa and Sven-Eric Lundgren Foundation,Crafoord Foundation, the Lundberg Foundation, the NovoNordisk Foundation, and the European Association for theStudy of Diabetes (Sankyo Pharma).

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